The large-scale, economic purification of proteins continues to present challenges for the biopharmaceutical industry. Therapeutic proteins are typically produced using engineered prokaryotic or eukaryotic cell lines to express proteins of interest from a recombinant plasmid containing the gene encoding the protein. The cell culture processes used for producing those therapeutic proteins are known to produce proteins with varying degree of heterogeneity with respect to process-related impurities and product-related substances. Product-related substances typically include charge variants, aggregates, fragments, or other protein product species derived from alternative post-translational modifications. Process-related impurities include, for example, host cell proteins (HCPs), DNA, endotoxin, virus and cell culture media components. Control over such process-related impurities and product-related substances can impact numerous product characteristics, including, but not limited to, product stability, product safety and product efficacy.
Although various techniques are available for large-scale protein purification, the separation of product-related substances, including charge variant species, remains challenging. For example, the charge variants in monoclonal antibody preparations typically include acidic, main and basic species, which can be detected by WCX-10 HPLC (a weak cation exchange chromatography) or IEF (isoelectric focusing). The very similar physio-chemical characteristics between the main protein species and the acidic and basic variant species require the use of highly selective separation systems and methods in order to achieve efficient separation.
Displacement chromatography is a chromatographic separation technology that involves the use of a displacer molecule to aid in the separation of a mixture, e.g., an antibody-containing solution derived from cell culture harvest. The displacer molecule is conventionally selected to have a higher affinity for the stationary phase (i.e., the chromatographic support) as compared to the components present in the material to be separated. Due to its higher affinity, the displacer molecule competes with protein mixture components for the binding sites on the stationary phase. Under appropriate conditions, the displacer induces the components of the mixture to develop into consecutive zones of concentrated and purified species in the order of decreasing binding affinity ahead of the displacer front. This ordered displacement of the components of the mixture results in the formation of a so-called “displacement train.” In contrast to traditional elution mode chromatography, the displacement process takes advantage of the nonlinearity of the adsorption isotherm, allowing for higher column loading levels without compromising the purity and recovery of the component of interest. Finally, washing of the displacement train with the displacing buffer from the column allows for the component of interest to be isolated by collecting (and pooling if necessary) the proper fraction(s) of the displaced eluate. Displacement chromatography in described, in general, in Brgles et al., Journal of Chromatography A, 1218 (2011) 2389-2395; Gajdosik et al., Journal of Chromatography A, 1239 (2012) 1-9; Gerstner et al., Biotechnol. Prog., (1992), 8, 540-545; Kundu et al., Analytical Biochemistry, 248, 111-116, (1997); and Vogt et al., Journal of Chromatography A, 760 (1997) 125-137.